EP3386540A1 - Nouvelles formulations d'adjuvant - Google Patents

Nouvelles formulations d'adjuvant

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Publication number
EP3386540A1
EP3386540A1 EP16808612.2A EP16808612A EP3386540A1 EP 3386540 A1 EP3386540 A1 EP 3386540A1 EP 16808612 A EP16808612 A EP 16808612A EP 3386540 A1 EP3386540 A1 EP 3386540A1
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EP
European Patent Office
Prior art keywords
composition
slns
crx
sln
agp
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP16808612.2A
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German (de)
English (en)
Inventor
David Burkhart
Jay T EVANS
Hardeep OBEROI
Yvonne M YORGENSEN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GlaxoSmithKline Biologicals SA
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GlaxoSmithKline Biologicals SA
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Publication of EP3386540A1 publication Critical patent/EP3386540A1/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7008—Compounds having an amino group directly attached to a carbon atom of the saccharide radical, e.g. D-galactosamine, ranimustine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/722—Chitin, chitosan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to novel adjuvant formulations and methods for the preparation thereof, as well as to uses thereof.
  • the invention relates to novel formulations of am inoalkyi glucosaminide phosphates (AGPs).
  • AGPs am inoalkyi glucosaminide phosphates
  • TLRs Toll-like receptors
  • LPS a bacterial membrane component specific to Gram-negative bacteria, which activates TLR4.
  • AGPs are a different class of compounds which interact with TLR4, as agonists or antagonists.
  • AGPs include both acyclic and cyclic compounds and have been described e.g. in US 6,1 13,918, US 6,303,347, WO 98/50399, WO 01134617, WO 01190129, WO 021 12258 and WO 2004062599. These compounds have been demonstrated to retain significant adjuvant characteristics when formulated with antigens in vaccine compositions.
  • AGPs also demonstrate mucosal adjuvant activity and are effective in the absence of antigen, making them attractive compounds for prophylactic and/or therapeutic use.
  • AGPs have improved toxicity profiles when compared with LPS or derivatives thereof, their use in pharmaceutical settings will be benefited by further improvement of the toxicity profile, including less pyrogenic formulations.
  • the invention relates to a composition comprising solid lipid nanoparticles (SLNs), wherein the SLNs comprise an aminoalkyl glucosaminide phosphate (AGP).
  • SSNs solid lipid nanoparticles
  • AGP aminoalkyl glucosaminide phosphate
  • the invention in a second aspect, relates to an immunogenic composition comprising an SLN composition as described herein and an antigen. In a further aspect, the invention relates to a composition as described herein, or an immunogenic composition as described herein, for use in medicine, such as for use in the immunisation of a human subject.
  • the invention relates to a method for preparing a composition comprising SLNs as described herein, said method comprising:
  • method optionally includes a filtration step.
  • FIG. 1 Cationic lipids/surfactants used in preparation of cationic SLNs
  • FIG. 2 Post-secondary serum IgG titers (top), post-tertiary serum IgG titers (middle) and post-tertiary tracheal/vaginal wash IgA titers (bottom; vaginal wash - black bars, tracheal wash - grey bars) from SLN CRX-601 formulation study-1 .
  • CAT SLN cationic SLNs, SLN-1 .5%: SLNs prepared at 1 .5% w/v of the carrier lipid, SLN-5%: SLNs prepared at 5% w/v of the carrier lipid.
  • the values 1 ig or 5 ig refer to CRX- 601 dosage/animal/vaccination.
  • Figure 3 Post-tertiary serum HAI titers from sublingual evaluation of SLN/chitosan CRX-601 formulations study-1 . All formulations tested were able to generate functional antibodies. The SLN-5%/CRX-601 and SLN/CRX-601 + chitosan groups were able to generate antibodies at levels similar to control IM group. Statistical analysis: One-way ANOVA analysis of variance and Tukey post test.
  • FIG. 4 Post-secondary serum IgG titers (top), post-tertiary serum IgG titers (middle) and post-tertiary tracheal/vaginal wash IgA titers (bottom) from SLN CRX-601 formulation study-2.
  • CAT SLN cationic SLNs, SLN-1 .5%: SLNs prepared at 1 .5% w/v of the carrier lipid, SLN-5%: SLNs prepared at 5% w/v of the carrier lipid.
  • CRX-601 dose is 5 g/animal/vaccination in all SL treatment groups and 1 g/animal/vaccination in the IM control.
  • FIG. 5 Post-tertiary serum HAI titers from sublingual evaluation of SLN/chitosan CRX-601 formulations study-2. All formulations tested were able to generate functional antibodies at levels generally similar to control IM groups (except Blk Cat SLN (SA-0.5%) old and BLK CAT SLN (SA-1 %). Statistical analysis: One-way ANOVA analysis of variance and Tukey post test.
  • solid lipid nanoparticle when used herein refers to a carrier in the submicron size range comprising lipids and surfactants and having a solidified lipid core.
  • the lipids are typically biocompatible and biodegradable and solid at room and body temperature.
  • AGP refers to.aminoalkyl glucosaminide phosphates. This application discloses a number of examples of AGPs and includes references to further documents describing AGPs. Even further AGPs and information on the synthesis of AGPs can e.g. be found in Johnson et al. (1999) Bioorg Med Chem Lett 9: 2273.
  • AGPs used in the present invention are preferably TLR4 agonists, i.e. compounds which activate a TLR4 receptor to produce a biological response.
  • mucus refers to a tendency for adhesion to mucus or mucus membranes.
  • Transmucosal administration refers to administration of a substance through mucus or mucus membranes.
  • the invention relates to a composition comprising solid lipid nanoparticles (SLNs), wherein the SLNs comprise an aminoalkyl glucosaminide phosphate (AGP).
  • SSNs solid lipid nanoparticles
  • AGP aminoalkyl glucosaminide phosphate
  • the AGP used in the invention is a TLR4 agonist.
  • AGPs suitable for use in the invention are disclosed in, for example, WO9850399, WO0134617, WO0212258, W03065806, WO04062599, WO06016997, WO0612425, WO03066065, WO0190129 and WO2012055981 , US 2003/0199460 and US 2005/0227943, the disclosure of which is herein incorporated by reference.
  • Such molecules have also been described in the scientific and patent literature as lipid A mimetics.
  • the AGP is one in which an aminoalkyl (aglycon) group is glycosidically linked to a 2-deoxy-2-amino-a-D- glucopyranose (glucosaminide).
  • the compounds are phosphorylated at the 4 or 6 carbon on the glucosaminide ring.
  • the compounds possess three 3-alkanoyloxyalkanoyl residues comprising a primary and secondary fatty acyl chain, each carbon chain consisting of from 2-24 carbon atoms, and preferably from 7-16 carbon atoms.
  • each primary chain contains 14 carbon atoms and each secondary chain has between 10 and 14 carbon atoms.
  • the AGP compounds are described by the general formula:
  • Such compounds comprise a 2-deoxy-2-amino-a-D-glucopyranose (glucosamine) in glycosidic linkage with an aminoalkyl (aglycon) group.
  • glucosamine 2-deoxy-2-amino-a-D-glucopyranose
  • aminoalkyl aminoalkyl
  • R1 , R2, and R3 represent normal fatty acyl residues having 7 to 16 carbon atoms
  • R4 and R5 are hydrogen or methyl
  • R6 and R7 are hydrogen, hydroxy, alkoxy, phosphono, phosphonooxy, sulfo, sulfooxy, amino, mercapto, cyano, nitro, formyl or carboxy and esters and amides thereof
  • R8 and R9 are phosphono or hydrogen.
  • the configuration of the 3' stereogenic centers to which the normal fatty acyl residues are attached is R or S, but preferably R.
  • the stereochemistry of the carbon atoms to which R4 or R5 are attached can be R or S.
  • X is oxygen.
  • the number of carbon atoms between heteroatom X and the aglycon nitrogen atom is determined by variables "n" and "m". Variables "n” and “m” can be integers from 0 to 6. In a preferred embodiment, the total number of carbon atoms between heteroatom X and the aglycon nitrogen atom is from about 2 to about 6 and most preferably from about 2 to about 4.
  • R8 is phosphono and R9 is hydrogen.
  • the compounds are hexaacylated, that is, they contain a total of six fatty acid residues.
  • the aminoalkyl glucosamine moiety is acylated at the 2- amino and 3-hydroxyl groups of the glucosamine unit and at the amino group of the aglycon unit with 3-hydroxyalkanoyl residues. In Formula I, these three positions are acylated with 3-hydroxytetradecanoyl moieties.
  • the 3-hydroxytetradecanoyl residues are, in turn, substituted with normal fatty acids (R1 -R3), providing three 3-n- alkanoyloxytetradecanoyl residues or six fatty acid groups in total.
  • the chain length of normal fatty acids R1-R3 can be from about 7 to about 16 carbons. Preferably, R1 -R3 are from about 9 to about 14 carbons.
  • the chain lengths of these normal fatty acids can be the same or different. Although only normal fatty acids are described, it is expected that unsaturated fatty acids (i.e. fatty acid moieties having double or triple bonds) substituted at R1 ,-R3 on the compounds would produce biologically active molecules.
  • AGPs suitable for use in the invention include CRX- 527 which is disclosed in Stover et al (2004) J Biol Chem 279, 6, page 4440.
  • WO0212258 and W03065806 disclose additional suitable embodiments of AGPs having a cyclic aminoalkyl (aglycon) linked to a 2-deoxy-2-amino-a-D- glucopyranose (glucosaminide), commonly referred to as "cyclic AGPs.”
  • cyclic AGPs a cyclic aminoalkyl (aglycon) linked to a 2-deoxy-2-amino-a-D- glucopyranose (glucosaminide)
  • Reference generally to AGPs herein includes both cyclic and non cyclic AGPs.
  • Cyclic AGPs possess three 3-alkanoyloxyalkanoyl residues comprising a primary and secondary fatty acyl chain, each carbon chain consisting of from 2-24 carbon atoms, and preferably from 7-16 carbon atoms. In one preferred aspect each primary chain contains 14 carbon atoms and each secondary carbon chain has between 10 and 14 carbon atoms per chain.
  • These compounds comprise a 2-deoxy-2-amino-p-D-glucopyranose (glucosamine) glycosidically linked to a cyclic aminoalkyl (aglycon) group.
  • the compounds are phosphorylated at the 4 or 6-position of the glucosamine ring and acylated with alkanoyloxytetradecanoyl residues on the aglycon nitrogen and the 2 and 3-positions of the glucosamine ring.
  • the compounds are described generally by formula (II): and pharmaceutically acceptable salts thereof, wherein X is-O-or NH-and Y is-O-or -S-; R1 , R2, and R3 are each independently a (C2-C24) acyl group, including saturated, unsaturated and branched acyl groups; R4 is -H or -PO3R7R8, wherein R7 and R8 are each independently H or (C1-C4) alkyl ; R5 is -H, -CH 3 or - PO3R9R10, wherein R9 and RIO are each independently selected from-H and (CI-C4) alkyl ; R6 is independently selected from H, OH, (CI-C4) alkoxy, -PO3R11 R12, -OPO3R11 R12, -SO3R1 1 , -OSO3R1 1 , - NR1 1 R12, -SR11 , -CN, -NO2, -CHO,
  • the cyclic AGP compound contains an- -O-at X and Y
  • R4 is PO3R7R8,
  • R5 and R6 are H
  • the subscripts n, m , p, and q are integers from 0 to 3.
  • R7 and R8 are -H.
  • subscript n is 1
  • subscript m is 2
  • subscripts p and q are 0.
  • R1 , R2, and R3 are tetradecanoyi residues.
  • *1-3 are in the R configuration, Y is in the equatorial position, and ** is in the S configuration (N-[(R)-3-tetradecanoyloxytetradecanoyl]-(S)-2- pyrrolidinomethyl 2-deoxy-4-0- phosphono-2-[(R)-3- tetradecanoyloxytetradecanoylamino]-3-0-[(R)-3-tetradecanoyloxytetradecanoyl]-p- D- glucopyranoside and pharmaceutically acceptable salts thereof).
  • Preferred cyclic structures include:
  • Formula V is CRX 590.
  • the AGP has one or more ether linked rather than ester linked primary and/or secondary lipid groups.
  • R1 -R3 represent straight chain alkyl groups and not acyl groups, making the groups R10-, R20-, and R30- alkoxy rather than alkanoyloxy groups and the attachment to the primary acyl chain an ether rather than an ester linkage.
  • composition comprises between, 0.1 and 1 .5% (w/v) of CRX-601 , more preferably between 0.1 and 1 %, even more preferably between 0.2 and 0.5%, such as between 0.2 and 0.25%, or between 0.5 and 1 %.
  • the AGP molecule may have a different number of carbons in the molecule's primary chains and/or secondary chains. Such compounds are disclosed in WO04062599 and WO06016997.
  • each carbon chain may consist of from 2-24 carbon atoms, and preferably from 7-16 carbon atoms.
  • each primary chain contains 14 carbon atoms and each secondary carbon chain has between 10 and 14 carbon atoms per chain.
  • Such compounds are represented by the following structures VII, VIII, IX and X:
  • X is selected from the group consisting of O and S at the axial or equatorial position; Y is selected from the group consisting of O and NH ; n and m are 0; R1 , R2 and R3 are the same or different and are fatty acyl residues having from 1 to about 20 carbon atoms and where one of R1 , R2 or R3 is optionally hydrogen; R4 is selected from the group consisting of H and methyl; p is 1 and R6 is COOH or p is 2 and R6 is OPO3H2 ; R8 and R9 are the same or different and are selected from the group consisting of phosphono and H, and at least one of R8 and R9 is phosphono; and R10, R11 and R12 are independently selected from straight chain unsubstituted saturated aliphatic groups having from 1 to 10 carbon atoms; or a pharmaceutically acceptable salt thereof.
  • X is selected from the group consisting of O and S at the axial or equatorial position; Y is selected from the group consisting of O and NH; n, m, p and q are integers from 0 to 6; R1 , R2 and R3 are the same or different and are straight chain saturated aliphatic groups (i.e., straight chain alkyl groups) having from 1 to about 20 carbon atoms and where one of R1 , R2 or R3 is optionally hydrogen; R4 and R5 are the same or different and are selected from the group consisting of H and methyl; R6 and R7 are the same or different and are selected from the group consisting of H, hydroxy, alkoxy, phosphono, phosphonooxy, sulfo, sulfooxy, amino, mercapto, cyano, nitro, formyl and carboxy, and esters and amides thereof; R8 and R9 are the same or different and are selected from the group consisting of phosphon
  • the general formula may also comprise an R5 group, at the same position as shown in formula VII above, wherein R5 is selected from the group consisting of H and methyl.
  • Yet another type of compound of this invention has the formula above (IV): wherein Y is now fixed as oxygen; X is selected from the group consisting of O and S at the axial or equatorial position; n and m are 0; R1 , R2 and R3 are the same or different and are fatty acyl residues having from 1 to about 20 carbon atoms and where one of R1 , R2 or R3 is optionally hydrogen; R4 is selected from the group consisting of H and methyl; p is 0 or1 and R6 is COOH, or p is 1 or 2 and R6 is OP03H2 ; R8 and R9 are the same or different and are selected from the group consisting of phosphono and H, and at least one of R8 and R9 is phosphono; and R10, R1 1 and R12 are independently selected from straight chain unsubstituted saturated aliphatic groups having from 1 to 10 carbon atoms; or a pharmaceutically acceptable salt thereof.
  • Lipids and surfactants suitable for use in the invention Lipids and surfactants suitable for use in the invention
  • SLNs are carriers in the submbron size range comprising lipids and surfactants and having a solidified lipid core.
  • the lipids used are typically biocompatible and biodegradable and solid at room and body temperature.
  • lipids can be used for SLN preparation, and examples of suitable lipids are e.g. given Table 8.1 of Svilenov and Tzachev (2014) Nanomedbine Chapter 8, page 187 (ISBN (eBook): 978-1 -910086-01 - 8: Publisher: One Central Press (OCP)) (herein incorporated by reference).
  • Preferred lipids for use in the present invention are behenates of glycerol, i.e. monobehenate of glycerol, dibehenate of glycerol and tribehenate of glycerol. Preferred are also mixtures of these three. Preferred concentrations are from 1 to 5% (w/v), such as from 1 to 4%, e.g. 1 .5% or 3%.
  • a preferred lipid is Compritol 888 ATO (glyceryl dibehenate European Pharmacopoeia [EP], glyceryl behenate National Formulary [NF]), which is a hydrophobic mixture of mono- (12 - 18% w/w), di- (45 - 54% w/w) and tri- (28 - 32% w/w) behenate of glycerol with melting point in range of 69 - 74°C and with hydrophilic lipophilic balance (HLB) « 2.
  • EP European Pharmacopoeia
  • NF National Formulary
  • surfactants can be used for SLN preparation, and examples of suitable surfactant are e.g. given Table 8.2 of Svilenov and Tzachev (2014) Nanomedicine Chapter 8, page 187 (ISBN (eBook): 978-1 -910086-01 -8: Publisher: One Central Press (OCP)) (herein incorporated by reference).
  • a preferred surfactant is polysorbate 80 (also known as Tween 80).
  • the composition comprises from 0.5 to 3% (v/v) polysorbate 80.
  • compositions of the invention are mucoadhesive.
  • SLNs can be rendered more mucoadhesive by combination with or incorporation of mucoadhesive compounds.
  • SLNs of the invention are cationic.
  • Cationic SLNs can e.g. potentially allow mucoadhesion by their electrostatic interaction with polyanionic mucin coating on the sublingual mucosa.
  • the Zeta potential e.g. as measured by dynamb light scattering is 20 mV or more, e.g. 20 to 30 mV, or more.
  • SLNs can be rendered more cationic by combination with or incorporation of cationic lipids and/or cationic surfactants.
  • the SLNs in the composition of the invention comprise a cationic lipid and/or a cationic surfactant.
  • the SLNs comprise a compound selected from the group consisting of stearylamine (Octadecylamine) (SA), dimethyldioctadecylammonium bromide (DDAB), or cetyltrimethylammonium bromide (CTAB).
  • SA stearylamine
  • DDAB dimethyldioctadecylammonium bromide
  • CTAB cetyltrimethylammonium bromide
  • the composition comprises 0.25-1 .5% SA, DDAB or CTAB, such as 0.25 - 0.55%, e.g. 0.5% SA, DDAB or CTAB.
  • the SLNs are rendered mucoadhesive by addition of a mucoadhesive agent.
  • Preferred mucoadhesive agents are chitosan derivates, such as alkylated chitosans.
  • a particularly preferred mucoadhesive agent is as methylglycol chitosan, which is preferably used in a concentration of 0.2 to 20 mg/ml, more preferably from 2 to 10, such as from 2 to 5 mg/ml.
  • compositions of the invention are further preferred properties of the compositions of the invention.
  • the composition of the invention has a size distribution below the sterile filterability limit, to avoid loss of material during sterile filtration.
  • the average size of the SLNs in the composition is between 30 and 200 nm, such as between 30 and 150 nm, such as between 30 and 100 nm.
  • the polydispersity index is less than 0.75, such as less than 0.5, such as less than 0.4. Polydispersity index and size may e.g. be measured using a Malvern Zetasizer.
  • the composition remains a stable, homogeneous, composition and does not become a semisolid gel for at least 24 hrs following its preparation.
  • the invention relates to a method for preparing a composition comprising SLNs as described herein, said method comprising:
  • the method may include a filtration step.
  • compositions of the invention may be used in medicine.
  • the compositions may be used on their own or in combinations with other substances, for instance in combination with an antigen for use in immunization.
  • compositions of the invention may be used in such methods.
  • compositions of the invention may also be used as adjuvants in vaccination.
  • the invention relates to an immunogenic composition
  • an immunogenic composition comprising an SLN composition as described herein and an antigen.
  • Vaccine preparation is generally described in Vaccine Design ("The subunit and adjuvant approach” (eds Powell M. F. & Newman M.J .) (1995) Plenum Press New York).
  • the compositions of the invention are suitable for combination with many different antigens, including e.g. proteins, nucleic acids and polysaccharides of various origins.
  • the antigen is an influenza antigen, such as haemagglutinin.
  • compositions or immunogenic compositions of the invention may be used to protect or treat a mammal by means of administering via systemic or transmucosal route. These administrations may include injection via the intramuscular, intraperitoneal, intradermal or subcutaneous routes; or via transmucosal administration to the oral/alimentary, respiratory, genitourinary tracts. Administration via a transmucosal route, such as sublingual administration is preferred.
  • the composition of the invention may be administered as a single dose, or multiple doses.
  • Subject doses of the composition described herein typically range from about 0.1 ig to 10mg of AGP per administration.
  • Preferred mucosal or local doses range from 1 g to 10 mg of AGP per administration, and most typically 10 g to 1 mg of AGP.
  • CRX-601 20% w/v
  • Compritol 888 ATO glyceryl behenate: a mixture of mono-, di-, and tri-behenate of glycerol; 5% w/v
  • THF tetrahydrofuran
  • Variable amounts of these stock solutions were added to a glass vial so as to obtain the desired concentrations of CRX-601 and Compritol 888 ATO in the final aqueous preparation (shown in Table 1 ).
  • the organic solvent was removed by evaporation on a rotary evaporator and further with high pressure vacuum for 12 hrs.
  • the CRX-601 lipid mix thus obtained was heated to >80 °C, and to it an aqueous solution containing Tween 80 (0.25 - 2% v/v) was added at the same temperature under high speed mixing (8000 - 12000 rpm).
  • the resultant oil- in-water emulsion was sonicated further (15 - 25 min) using a probe sonicator on a hot plate maintained at a temperature >80 °C.
  • the SLN composition was then allowed to congeal at room temperature and aseptically filtered using a 0.22 ⁇ filter into a sterile depyrogenated container. Representative average particle sizes and zeta-potential values of the resultant formulations as measured by dynamic light scattering are shown in Table 1 .
  • CRX-601 used in this work was synthesized as described previously (Bazin et al. (2008) Bioorg & Med Chem Lett 18: 5350 (CRX-601 is the ether analog of CRX-527)) and purified by chromatography (to >95% purity). CRX-601 , either in the starting material or in the final product was quantified by a standard reverse phase HPLC analytical method.
  • Formulations with higher than 5 % w/v Compritol and > 1 % w/v CRX-601 yield nanoparticles with a size distribution approaching the sterile filterability limit, and are associated with loss of material during sterile filtration.
  • Table 1 Representative formulation parameters for CRX-601 SLNs.
  • Stearic acid was explored for preparation of SLNs (instead of glyceryl behenate). Stearic acid concentrations of 1 .5%, 3.0% and 6.0% were used. CRX-601 was targeted at 1 , 2 and 3mg/mL. Clear homogeneous formulations were initially seen right after preparation, but upon storage ovemight turned into semisolid gels.
  • SLN formulations based on glyceryl behenate are anionic in zeta-potential.
  • Cationic SLNs were prepared by incorporation of cationic lipids/surfactants (shown in Figure 1 ) into the glyceryl behenate SLNs.
  • the cationic SLNs could potentially allow mucoadhesion by their electrostatic interaction with polyanionic mucin coating on the sublingual mucosa.
  • SA 0.1 - 1 % w/v stearylamine
  • DDAB dimethyldioctadecylammonium bromide
  • CTAB cetyltrimethylammonium bromide
  • Methylglycol chitosan (chitosan glycol trimethyl ammonium iodide) was dissolved in 10 mM HEPES or 10 mM HEPES-saline buffer pH 7.2 to yield concentrations of 2 and 10 mg/ml.
  • the solutions were asceptically filtered using a 0.22 ⁇ filter into a sterile depyrogenated container.
  • the formulations from Examples 1 and 2 were mixed aseptically with varying volumes of methylglycol chitosan solution to yield concentrations of methylglycol chitosan ranging from 1 - 10 mg/mL. Representative average particle sizes and zeta-potential as measured by dynamic light scattering are shown in Table 3.
  • sonication of the formulation on a water bath sonicator for 10min was used to reduce PDI values and make the size distribution unimodal.
  • the data indicates some increase in particle size and a reversal in zeta-potential (net positive potential from a net negative potential) exceeding approximately 1 mg/mL methylglycol chitosan, consistent with surface coating with methylglycol chitosan.
  • zeta-potential net positive potential from a net negative potential
  • Table 3 Summary of formulation parameters for methylglycol chitosan coated SLN-CRX-601.
  • NT Not tested, U: Unimodal, B: Bimodal, M: Multimodal
  • Table 4 Summary of formulation parameters for methylglycol chitosan associated SLNs prepared using method of Sandri et al.
  • the pyrogen test is used here as a surrogate measure of CRX-601 incorporation into SLNs from Example 1 - 3 and as a measure of their stability in biological milieu.
  • the test was performed at Pacific Biolabs (Hercules, CA) as per their SOP 16E-02, which follows procedures outlined in USP ⁇ 151 >.
  • the 1.5% SLN/CRX-601 formulation lacked pyrogenicity at a concentration of 25 ng CRX-601/kg animal body weight. This lack of pyrogenicity up to 25 ng/kg corresponding to an at least 10 fold improvement over free CRX- 601 (max non-pyrogenic dose of 1 -2 ng/kg), and indicates a >90% incorporation of CRX-601 into the SLNs.
  • the individual temperature increases from three rabbits per test are indicated in table 5.
  • Table 5 Representative rabbit pyrogen test measurements for some of the formulations described in Example 1 , 2, and 3. Values in parenthesis are maximum temperature change for three animals during the testing period.
  • CRX-601 dose 2.5 CRX-601 dose: 25 CRX-601 dose: 50 ng/kg ng/kg ng/kg
  • CRX-601 /3%-SLN NP 0.0, 0.1 , 0.0
  • NP 0.0, 0.2, 0.3
  • P 0.6, 0.6, 0.7
  • NT not tested; *NP: Non-pyrogenic response, P: Pyrogenic response.
  • the test is considered NP if none of the rabbits shows an individual temperature rise of 0.5 °C or more above its respective control temperature at any point.
  • Example 6 Mouse sublingual vaccination and determination of specific antibody responses
  • mice Female BALB/c mice (6 to 8 weeks of age) obtained from Charles River Laboratories (Wilmington, MA) were used for these studies. Mice anesthetized by intraperitoneal (i.p) administration of ketamine (100 mg/kg) and xylazine (10 mg/kg) were given vaccine by sublingual administration (5 - 6 ⁇ ). All mice were vaccinated on days 0, 21 and 42 with the 5 ig CRX-601 in the SLN formulation admixed with 1 or 1 .5 ig HA/mouse using the influenza antigen A/Victoria/210/2009 H3N2.
  • ELISA was performed using split flu coated 96 well plates (Nunc Maxisorp) and detecting the bound immunoglobins from the added serum or tracheal wash or vaginal wash samples using peroxidase linked goat anti-mouse IgG, lgG1 , lgG2a or IgA. This was followed by addition of an enzyme specific chromogen, which resulted in color intensity directly proportional to the amount of specific antiflu IgGs/lgAs contained in the serum. The optical density was read at 450 nm.
  • HI assay was performed by evaluating inhibition of chicken or rooster RBCs upon exposure to flu virus in presence of mouse serum. The reciprocal of the last dilution of influenza virus which resulted in complete or partial agglutination of RBCs was used to calculate the HI titer and expressed as HA units / 50 ⁇ of sera.
  • Example 7 Mouse sublingual vaccination with SLN formulations (NIH # 157, 165, 170, 171)
  • Table 6 Summary of formulations prepared for mouse sublingual study (study-1 ).
  • mice were vaccinated using the procedure outlined in Example 6 with formulations from table 6.
  • Post- secondary serum antibody titers were higher with SLN formulations of CRX-601 compared to vehicle controls or a CRX-601 DOPC (1 ,2-dioleoyl-sn-glycero-3-phosphocholine) liposome formulation (figure 2, top panel), with the exception of cationic SLN CRX-601 , which was not better than the controls in this particular assay.
  • Mice vaccinated via intramuscular administration of CRX-601 + split flu antigen are also included as a positive control.
  • Serum HI titers were highest in mice vaccinated with chitosan coated 1.5%-SLN 5 ig CRX-601 group and significantly higher than the CRX-601 DOPC liposome group (figure 3). No significant differences were noted between other SL treatment groups.
  • the follow up study includes additional groups with blank cationic SLNs, additional ratio of SLN-1 .5%/CRX-601 + MGC, and additional group with SLN- 5%/CRX-601 + MGC formulation compositions.
  • Table 7 SLN formulations prepared for mouse sublingual study-2. All formulations were prepared at a CRX-601 concentration > 1 img/mL and were further diluted to 1 img/mL before transfer to immunology. Blank
  • SLN formulations particularly with 5-10 img/mL chitosan, are promising lead candidates for SL vaccination formulations.
  • CAT SLN formulations without CRX- 601 elicited significant levels of IgG and IgA.
  • Post-tertiary HI titers corresponded to post-tertiary IgG titers. All formulations tested were able to generate functional antibodies at levels generally similar to control IM groups (except BIk CAT SLN (SA- 0.5%) old and BLK CAT SLN (SA-1 %) (figure 5).

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Abstract

L'invention concerne une composition comprenant des nanoparticules lipidiques solides (SLN), lesdites SLN comprenant un aminoalkylglucosaminide phosphate (AGP).
EP16808612.2A 2015-12-08 2016-12-06 Nouvelles formulations d'adjuvant Withdrawn EP3386540A1 (fr)

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